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The effective binding of anions like carboxylates and phosphates in aqueous solutions is of particular interest for various reasons. The natural archetypes of effective anion receptors are enzymes that contain often arginine as relevant amino acid in the binding pocket. For this reason, one class of artificial anion receptors that emerged more than two decades ago mimics the anion binding with the guanidinium group present in the amino acid side chain. In 1999, Schmuck and coworkers developed a new class of guanidinium-based oxo anion receptor that binds carboxylates even in aqueous media. The binding modes of the 2-(guanidiniocarbonyl)-1H-pyrroles are based on individually weak non-covalent interaction between artificial host and substrate like ion pairing and multiple hydrogen bonds. The zwitterionic derivative with substitution of a carboxylate group in position 5 of the pyrrole ring system shows a strong self-assembly to discrete dimers (dimer 1) with an estimated association constant of 170 M-1 even in water. In order to further improve the structure motif for an effective oxo anion binding it is therefore of great interest to quantify the different intermolecular interactions between two monomeric units of 1. Against this background several theoretical ab initio studies were conducted in order to elucidate the influences of intrinsic properties as well as solvent effects on the stability of self-assembled dimers. In chapter 4.1 the molecular interactions in dimer 1 were investigated by comparison to various “knock-out” analogues. In these analogues single hydrogen bonds were switched off by substitution of hydrogen donor atoms with either methylene groups or ether bridges. The calculations were done for vacuum and solvation, as represented by a conductor-like polarizable continuum. It could be shown that the application of a simple continuum solvent model fails to predict the absolute energies of the knock-out analogues in strongly polar solvents. However, the calculated trends can explain the relative stabilities. In chapter 4.2 the structural similarity of arginine with structure 1 was used in order to examine the dependence of self-assembly from the flexibility of the molecular structure. In chapter 4.2.1 new global minimum structures of the canonical and zwitterionic arginine in gas phase were found by means of exhaustive force field based conformational searches in conjunction with ab initio structure optimizations of the lowest energy conformers. Most of the newly identified minimum conformers of both the zwitterionic and canonical tautomer revealed geometrical arrangements with hitherto unreported stacked orientations of the terminal groups. Finally a novel global minimum structure was detected that is more than 8 kJ mol-1 lower in energy than the previously published conformers. The same strategy for finding minimum energy conformers of the arginine monomer has also been employed for the arginine dimer structures. While previous theoretical studies favoured directed hydrogen bonds the new global minimum structure MMFF1 is about 60 kJ mol-1 more stable and exhibits a stacked orientation of the guanidinium and carboxylate groups. The importance of rigidity on the dimer stability was proven by calculations of an artificially stiffened arginine dimer system. The high binding affinity dimer 1 results by about 50% from the rigidity of the monomers which prevents any intramolecular stabilization. In chapter 4.3 novel structure motifs with varying ring systems have been examined on a DFT level of theory in order to make proposals for an improved carboxylate binding motif. The direct dependency of the dimerization energy on an increasing dipole moment was demonstrated by various anellated ring structures. The influence of the delocalization in the monomer on the dimerization energy was examined by variation of the electronic structure of electronically decoupled biphenylenes. With the aid of various substituted 7-guanidinioindole-2-carboxylate derivatives we could show that the carbonyl function is mainly responsible for the advantageous preorganisation, whereas the effect on the acidity seems to be only of minor importance. In the last chapter cooperativity effects in supramolecular assemblies have been investigated. This was achieved by NMR shift calculations of adenosine-carboxylic acid complexes as model systems and comparison to experimental low-temperature NMR studies. We could demonstrate that only by applying vibrational averaged NMR shifts the experimental proton shifts obtained at very low temperatures in the hydrogen bond exchange regime could be reproduced.
The continuously increase in resistance of human pathogenic microorganisms to the known antibiotics leads to the necessity for searching new sources for production of new active antimicrobial compounds from different natural sources especially plants, since many plants have been found to be able to produce antimicrobial compounds as a defense phenomenon against invading microorganisms. The aim of this work is to screen cultures for production of antimicrobial activity against representative of human pathogenic microorganisms and selection the most active cell culture producing antimicrobial protein(s) which are active against these pathogenic microorganisms and also isolation ,purification of the active protein(s) and cloning of its/their genes. Ten different plant suspension cultures have been screened in presence of nine elicitors for their antimicrobial activity against five selected human pathogenic microorganisms, and it has been found that the heterotrophic cultures are more active against the tester isolates than the autotrophic ones. The intracellular fraction of the mixotrophic Arabidopsis thaliana culture elicited with salicylic acid showed the highest antimicrobial activity against the tester isolates. The presence of proteinous antimicrobial activity has been elucidated by testing the activity of ammonium sulphate precipitate against Candida maltosa. High speed centrifugation technique has been used for partial purification of the active protein. The proteinous nature of the isolated compound has been confirmed by using bioautography technique and its molecular weight could be estimated to be around 26KDa. The active protein has been purified using gel filtration, and using mass spectrometry technique, for microsequencing of the active protein, it has been found that the function of the protein is unknown and we have termed it as AtPDP1 according to Arabidopsis thaliana Plat-Domain Protein1, since it contains a plant stress domain termed PLAT domain. It has been found that a second protein from the same plant with high homology level to AtPDP1 with the same domain, we termed it as AtPDP2. Genes for AtPDP1 and AtPDP2 have been cloned in E. coli using PGEM-T easy vector. The expression of both genes have been tested using Digital Northern program, and it has been observed that both genes are induced by different pathogens, chemicals known to induce defense in plant cells and also different hormones. We tried to clone the gene for AtPDP1 in PBI121 binary vector under the control of an elicitor inducible promoter of a proteinase inhibitor gene, to test its function in plant by overexpression, but we did not succeeded. Also the work aims to cloning the different known thaumatin genes from Arabidopsis thaliana for future work which represented by testing their expression under different stimuli, since most thaumatins have antimicrobial activity and some of them are active against Candida spp..Thirteen genes of known thaumatins from Arabidopsis thaliana have been cloned in PGEM-Teasy vector in DH5-alpha cells. coli cells. The expression of the thirteen genes has been done using Digital Northern program and it has been found that different genes show different expressions under different stimuli and the expression of At1g75800 gene was the maximum under all stimuli. The minimum expression of genes was for At1g75050. The rest of thaumatin genes showed moderate expressions under different stimuli.
Despite its precise agreement with the experiment, the validity of the standard model (SM) of elementary particle physics is ensured only up to a scale of several hundred GeV so far. Even more, the inclusion of gravity into an unifying theory poses a problem which cannot be solved by ordinary quantum field theory (QFT). String theory, which is the most popular ansatz for a unified theory, predicts QFT on noncommutative space-time as a low energy limit. Nevertheless, independently of the motivation given by string theory, the nonlocality inherent to noncommutative QFT opens up the possibility for the inclusion of gravity. There are no theoretical predictions for the energy scale Lambda_NC at which noncommutative effects arise and it can be assumed to lie in the TeV range, which is the energy range probed by the next generation of colliders. Within this work we study the phenomenological consequences of a possible realization of QFT on noncommutative space-time relying on this assumption. The motivation for this thesis was given by the gap in the range of phenomenological studies of noncommutative effects in collider experiments, due to the absence in the literature of Large Hadron Collider (LHC) studies regarding noncommutative QFTs. In the first part we thus performed a phenomenological analysis of the hadronic process pp -> Z gamma -> l^+l^- gamma at the LHC and of electron-positron pair annihilation into a Z boson and a photon at the International Linear Collider (ILC). The noncommutative extension of the SM considered within this work relies on two building blocks: the Moyal-Weyl star-product of functions on ordinary space-time and the Seiberg-Witten maps. The latter relate the ordinary fields and parameters to their noncommutative counterparts such that ordinary gauge transformations induce noncommutative gauge transformations. This requirement is expressed by a set of inhomogeneous differential equations (the gauge equivalence equations) which are solved by the Seiberg-Witten maps order by order in the noncommutative parameter Theta. Thus, by means of the Moyal-Weyl star-product and the Seiberg-Witten maps a noncommutative extension of the SM as an effective theory as expansion in powers of Theta can be achieved, providing the framework of our phenomenological studies. A consequence of the noncommutativity of space-time is the violation of rotational invariance with respect to the beam axis. This effect shows up in the azimuthal dependence of cross sections, which is absent in the SM as well as in other models beyond the SM. Thus, the azimuthal dependence of the cross section is a typical signature of noncommutativity and can be used in order to discriminate it against other new physics effects. We have found this dependence to be best suited for deriving the sensitivity bounds on the noncommutative scale Lambda_NC. By studying pp -> Z gamma -> l^+l^- gamma to first order in the noncommutative parameter Theta, we show in the first part of this work that measurements at the LHC are sensitive to noncommutative effects only in certain cases, giving bounds on the noncommutative scale of Lambda_NC > 1.2 TeV. Our result improved the bounds present in the literature coming from past and present collider experiments by one order of magnitude. In order to explore the whole parameter range of the noncommutativity, ILC studies are required. By means of e^+e^- -> Z gamma -> l^+l^- gamma to first order in Theta we have shown that ILC measurements are complementary to LHC measurements of the noncommutative parameters. In addition, the bounds on Lambda_NC derived from the ILC are significantly higher and reach Lambda_NC > 6 TeV. The second part of this work arose from the necessity to enlarge the range of validity of our model towards higher energies. Thus, we expand the neutral current sector of the noncommutative SM to second order in $\theta$. We found that, against the general expectation, the theory must be enlarged by additional parameters. The new parameters enter the theory as ambiguities of the Seiberg-Witten maps. The latter are not uniquely determined and differ by homogeneous solutions of the gauge equivalence equations. The expectation was that the ambiguities correspond to field redefinitions and therefore should vanish in scattering matrix elements. However, we proved that this is not the case, and the ambiguities do affect physical observables. Our conjecture is, that every order in Theta will introduce new parameters to the theory. However, only the experiment can decide to what extent efforts with still higher orders in Theta are reasonable and will also give directions for the development of theoretical models of noncommutative QFTs.
Oxylipins are important biological active compounds that play essential roles in defense, growth, development, and reproduction of plants and animals. Oxylipins are formed either by enzymatic pathways or radical catalyzed reaction from polyunsaturated fatty acids. Products of oxidation of arachidonic acid (C20:4) in animals by enzymatic and non-enzymatic pathways are prostaglandins and isoprostanes, respectively. In plants, radical catalyzed reaction of -linolenic acid (C18:3) forms phytoprostanes and enzymatic oxidation of this fatty acid produces OPDA and jasmonic acid. Like plants, cyanobacterial membranes contain a high ratio of polyunsaturated fatty acid, about 25% of total fatty acids. Oxylipin biosynthesis and function was studied in two model cyanobacteria, Anabaena PCC 7120 and Synechocystis PCC 6803, for the first time: 1. The filamentous cyanobaterium Anabaena PCC 7120 can naturally produce phytoprostanes type I and II as well as hydroxy fatty acids like in plants but lacks the enzymatic capacity to form jasmonates (12-oxo-phytodienoic acid and jasmonic acid) and prostaglandins. Data obtained provide the first evidence for the occurence of phytoprostanes in cyanobacteria as well as in the baterial kingdom. 2. By GC-MS analysis, the E1- and F1-phytoprostanes in Anabaena PCC 7120 were detected both in free and esterified form. Their levels are comparable with those in plants, in the range of ng/g DW. In one week old cultures, there was no evidence of PPF1 in the medium but its level accumulated up to 142 ng/l in six weeks old cultures. In contrast, PPE1 was stable over time, about 20 ng/g DW. Free cellular PPE1 was found about 4 times higher than that of PPF1, 80.5  23.6 and 24.1  10.9 ng/g DW, respectively. However, there was no significant difference in the total cellular levels of PPF1 and PPE1, ranging from 150 to about 200 ng/g DW. 3. Phytoprostanes are inducible in Anabaena. In the combination of oxidative stress (200 µM H2O2 or 10 µM CuSO4) with high light intensity (330 µE.m-2.s-1) for 8 h, levels of total cellular PPE1 and PPF1 were increased about 2 to 4 times. Interestingly, unlike in higher plants, application of oxidative stress or high light intensity alone showed no phytoprostaneous induction in this cyanobacterium. 4. When Anabaena cells were treated with phytoprostanes, Anabaena cells became remarkably resistant against subsequently applied – otherwise lethal – oxidative stress. All phytoprostanes displayed a high protective effect except for PPE1. The highest protection level was contributed by a mixture of PPA1 type I and II. After preincubation of Anabena cells with 100 µM PPA1–type I/II for 16 h followed by application of 1 mM H2O2 or 50 µM CuSO4 for 5 h, A1-phytoprostane pre-treatment protected 84.2% and 77.5% of the cells from cell death, respectively. Without oxylipins pre-treatment, about 98% of the cells were dead. Surprisingly, preincubation of Anabaena with other oxylipins derived from enzymatic pathway in plants and animals showed also an effect, however, the protection effect was low and ranged from 10 to 30%. In contrast, phytoprostanes did not protect Pseudomonas syringae and Escherichia coli from the toxicity of hydrogen peroxide. However, these bacteria do not synthesize polyunsaturated fatty acids and are therefore devoid of and not exposed to endogenously formed oxidized lipids. 5. Exogenous application of 100 µM PPF1 or 1.5 mM H2O2 for 90 min did not activate the expression of isiA in Anabaena. Oxylipins also displayed no effect on shinorine and tocopherol levels in Anabaena. However, application of 100 µM PPF1 for 6 h altered the protein expression in Anabaena. Most PPF1-modulated proteins are down-regulated and related to photosynthesis. Since oxidative stress only in combination with high light intensity increased lipid peroxidation, down-regulation of photosynthesis after recognition of oxidised lipids (phytoprostanes) may be a survival strategy of Anabaena to avoid damage by peroxidized lipids. 6. Dead plants may be the main source of (exogenous) phytoprostanes in the natural environment of Anabaena. Dry hay releases PPE1 and PPF1 (11 µg/g DW) into an aqueous environment. Anabaena is the typical cyanobacterium in paddy rice fields. After harvesting, most of uneconomical parts of rice plants are abundant on the field, which may release phytoprostanes that in turn might have an impact on cyanobacteria in the rice ecosystems. However, field research is needed to clarify this suspection. 7. A new class of oxylipins, phytoprostanes type III and IV, was identified and quantified in vitro. The two main phytoprostanes, PPE1 and PPF1 (type III and IV), can be obtained by autoxidation of -linolenic acid or Borage oil (containing 25% esterified -linolenic acid). After 12 days of autoxidation and subsequent hydrolysis, 1 g of Borage oil yielded 112.71 ± 1.93 µg of PPF1 and 3.80 ± 0.14 mg of PPE1. PPB1 and PPA1 (type III and IV) were prepared by isomerization and dehydration of PPE1 (type III and IV). The overall yield of PPB1 was 1.71 ± 0.04 mg/g oil (type III) and 2.09 ± 0.12 mg/g oil (type IV). Those of PPA1 were 8.38 ± 0.35 µg/g and 10.18 ± 0.30 µg/oil, respectively. 8. A rapid HPLC-MS/MS method for phytoprostane and phytohormone analysis has been developed. This method was applied to quantify free and esterified E1- and F1-phytoprostanes type III and IV in Synechocystis PCC 6803. The in vivo phytoprostanes type III and IV are present both in free and esterified form. The total cellular level of PPE1 type III and IV in Synechocystis is at least 2 times higher than that of PPF1. Unlike Anabaena, PPE1 and PPF1 were detectable in the medium of one week old Synechocystis cultures. Free levels of PPF1 in the medium (231.8 ± 36.2 ng/l) and in the cells (164.9 ± 15.2 ng/g DW) are lower than those of PPE1 (1003.3 ± 365.2 ng/l and 2331.0 ± 87.7 ng/g DW).
With ageing, the loss of bone mass correlates with the expansion of adipose tissue in human bone marrow thus facilitating bone-related diseases like osteopenia and osteoporosis. The molecular mechanisms underlying these events are still largely unknown. Reduced osteogenesis and concurrently enhanced adipogenesis might not only occur due to the impairment of conventional osteogenic differentiation originating from mesenchymal stem cells (MSCs). Additionally, transdifferentiation of (pre-)osteoblasts into adipocytes could contribute to the fatty conversion. Therefore, the aim of the present study was to prove the existence of transdifferentiation between the adipogenic and osteogenic lineage and to elucidate molecular mechanisms underlying this phenomenon. At first, a cell culture system of primary human MSCs was established that allowed for differentiation into the adipogenic and osteogenic lineage and proved that the MSC-derived adipocytes and pre-osteoblasts were capable of transdifferentiation (reprogramming) from one into the other lineage. Thereby, lineage-specific markers were completely reversed after reprogramming of pre-osteoblasts into adipocytes. The osteogenic transdifferentiation of adipocytes was slightly less efficient since osteogenic markers were present but the adipogenic ones partly persisted. Hence, plasticity also reached into the differentiation pathways of both lineages and the better performance of adipogenic reprogramming further supported the assumption of its occurrence in vivo. The subsequent examination of gene expression changes by microarray analyses that compared transdifferentiated cells with conventionally differentiated ones revealed high numbers of reproducibly regulated genes shortly after initiation of adipogenic and osteogenic reprogramming. Thereof, many genes were correlated with metabolism, transcription, and signal transduction as FGF, IGF, and Wnt signalling, but only few of the established adipogenesis- and none of the osteogenesis-associated marker genes were detected within 24 h after initiation of transdifferentiation. To find possible key control factors of transdifferentiation amongst the huge amount of regulated genes, a novel bioinformatic scoring scheme was developed that ranked genes due to their potential relevance for reprogramming. Besides the reproducibility and level of their regulation, also the possible reciprocity between the adipogenic and osteogenic transdifferentiation pathway was taken into account. Fibroblast growth factor 1 (FGF1) that ranked as one of the leading candidates to govern reprogramming was proven to inhibit adipogenic differentiation as well as adipogenic transdifferentiation in our cell culture system. Further examination of the FGF signalling pathway and other highly ranked genes could help to better understand the age-related fatty degeneration at the molecular level and therefore provide target molecules for therapeutic modulation of the plasticity of both lineages in order to inhibit adipogenic degeneration and to enhance osteogenesis.
The Nuclear Factors of Activated T cells (NFATs) are critical transcription factors that direct gene expression in immune and non-immune cells. Interaction of T cells with Ag-presenting cells results in the clustering of T-cell antigen receptor (TCR), co-receptors and integrins. Subsequent signal transduction resulting in NFAT activation leads to cytokine gene expression. Among the NFATs expressed in T cells, NFATc1 shows a unique induction property, which is essential for T cell differentiation and activation. It was revealed before that 3 major isoforms of NFATc1 are generated in activated T cells – the inducible short NFATc1/A, and the longer isoforms NFATc1/B and C. However, due to alternative splicing events and the existence of two different promoters and two alternative polyadenylation, we show here that 6 isoforms are synthesized in T cells which differ in their N-terminal and C-terminal peptides. In these experiments, we have identified these 6 isoforms by semi-quantitative long distance RT-PCR in several T cells subsets, and the inducible properties of 6 isoforms were investigated in those cells. The short NFATc1/A which is under control of the P1 promoter and the proximal pA1 polyadenylation site was the most prominent and inducible isoform in T effector cells. The transcription of the longer NFATc1/B and C isoforms is constitutive and even reduced in activated T lymphocytes. In addition to NFATc1 autoregulation, we tried to understand the NFATc1 gene regulation under the control of PKC pathways by microarray analysis. Compared to treatment of T cells with ionomycin alone (which enhances Ca++ flux), treatment of cells with the phorbolester TPA (leading to PKC activation) enhanced the induction of NFATc1. Microarray analysis revealed that PKC activation increased the transcription of NF-B1, Fos and JunB, which are important transcription factors binding to the regulatory regions of the NFATc1 gene. Besides the promoting effect of these transcription factors, we provided evidence that p53 and its targeting gene, Gadd45, exerted a negative effect on NFATc1 gene transcription. Summarizing all these results, we drew novel conclusions on NFATc1 expression, which provide a more detailed view on the regulatory mechanisms of NFATc1 transcription. Considering the high transcription and strong expression of NFATc1 in various human lymphomas, we propose that similar to NF-B, NFATc1/A plays a pivotal role in lymphomagenesis.
Cardiovascular disease is the major cause of mortality morbidity in both men and women in industrialized countries. The incidence of cardiovascular diseases in pre-menopausal women is lower compared to age-matched men but the risk of heart diseases increases dramatically after the onset of menopause.Therefore, it has been postulated that female sex hormones play an important role in cardiovascular health in pre-menopausal women. In contrast to clinical data, which failed to show positive estrogen effects on cardiovascular system of post- menopausal women, extensive experimental studies indicated cardioprotective effects of estrogens in laboratory animals. The majority of experimental estrogen substitution studies were performed with young individuals, thus the effects of ageing remain neglected and are poorly understood. The present project is the first attempt to study the cardiac effects of each estrogen receptor isoform (estrogen receptor alpha (ERa) and estrogen receptor beta (ERb)) in adult (“menopausal”) and senescent (“post- menopausal”) hypertensive rats. The female senescent spontaneously hypertensive rats (SHR) served as a model system for age- associated hypertension in females whereas young individuals were used for control experiments. Young and senescent SHR rats were treated with 17b- estradiol as well as new estrogen receptor isoform selective ligands 16a-LE2 (ERa agonist) and 8b-VE2 (ERb agonist). The results showed different functions of both estrogen receptor isoforms in cardiovascular system: ERa attenuated cardiac hypertrophy but not hypertension whereas ERb could significantly reduce both, blood pressure and cardiac hypertrophy. Surprisingly, both agonists and 17b- estradiol were effective in young animals but not in senescent SHR rats. These findings match with the clinical data and could be related to altered estrogen metabolism in senescent rats, since estrogen plasma levels did not increase to measurable extent in senescent animals receiving estrogen. Estrogen is metabolized by several 17b- hydroxysteroid dehydrogenase isoforms. In the current study, 17b- hydroxysteroid dehydrogenase type 10 (17b- HSD10) was identified as a novel protein- protein interaction partner of estrogen receptor alpha ligand binding domain (ERaLBD) in human heart. Cellular localization experiments of ERa in the cardiac myocytes showed nuclear and cytosolic localization pattern which overlapped partially with that of cardiac mitochondria. 17b-HSD10 is localized only in mitochondria. Direct interaction of both proteins was confirmed by pull- down experiments where 17b-HSD10 could be co-precipitated with ERa. Interestingly, protein interaction could be detected only under estrogen- free conditions whereas the presence of estrogen in the system blocked this interaction. Enzymatic assay which was developed in our laboratory, helped to define functional relevance of this interaction. The data obtained from enzymatic assays and protein- protein interaction studies strongly suggest that estrogen receptor could play an important role in the control of intracellular (or mitochondrial) estogen metabolism. The second potential ERa interaction partner in the heart- bladder cancer associated protein 10 (BLCAP10) - was initially identified in non- invasive bladder cancer cell lines. BLCAP10 protein expression in the heart as well as its localization pattern in cardiac myocytes is shown in the last part of the theses. Due to perinuclear localization similarity with ERb, we conclude that BLCAP10 could interact with ERb rather than with ERa. Poor BLCAP10 protein overexpression and toxicity in both, bacteria and eukaryotic cells, suggested that BLCAP10 could be involved in cell- cycle and/ or protein expression control. In summary, the results showed that isoform selective activation of estrogen receptors exert divergent effects in the cardiovascular system both by upregulation of aMHC expression or by lowering blood pressure. Hormones were effective in young animals but had only minor effects in senescent rats. The new ERa protein- protein interaction partners identified during the project provide new information about estrogen receptor function in the heart and its possible role in the regulation of estrogen homeostasis.
The internal transcribed spacer 2 (ITS2) of the ribosomal gene repeat is an increasingly important phylogenetic marker whose RNA secondary structure is widely conserved across eukaryotic organisms. The ITS2 database aims to be a comprehensive resource on ITS2 sequence and secondary structure, based on direct thermodynamic as well as homology modelled RNA folds. Results: (a) A rebuild of the original ITS2 database generation scripts applied to a current NCBI dataset reveal more than 60,000 ITS2 structures. This more than doubles the contents of the original database and triples it when including partial structures. (b) The end-user interface was rewritten, extended and now features user-defined homology modelling. (c) Other possible RNA structure discovery methods (namely suboptimal and shape folding) prove helpful but are not able to replace homology modelling. (d) A use case of the ITS2 database in conjunction with other tools developed at the department gave insight into molecular phylogenetic analysis with ITS2.
This work studies the convergence of trajectories of gradient-like systems. In the first part of this work continuous-time gradient-like systems are examined. Results on the convergence of integral curves of gradient systems to single points of Lojasiewicz and Kurdyka are extended to a class of gradient-like vector fields and gradient-like differential inclusions. In the second part of this work discrete-time gradient-like optimization methods on manifolds are studied. Methods for smooth and for nonsmooth optimization problems are considered. For these methods some convergence results are proven. Additionally the optimization methods for nonsmooth cost functions are applied to sphere packing problems on adjoint orbits.
The live sciences currently undergo a paradigm shift to computer aided discoveries. Discoveries in the live sciences were historically made by either direct observation or as a result of chemical assays. Today we see a growing shift toward computer aided analysis and visualization. This gradual process happens in microscopy. Multidimensional laser scanning microscopy can acquire very complex multichannel data from fixed or live specimen. New probes such as visible fluorescent proteins let us observe the expression of genes and track protein localization. Ion sensitive dyes change intensity with the concentration of ions in the cell. The laser scanning confocal allows us to record these processes in three dimensions over time. This work demonstrates the application of software analysis to multidimensional microscopy data. We introduce methods for volume investigation, ion flux analysis and molecular modeling. The visualization methods are based on a multidimensional data model to accommodate complex datasets. The software uses vector processing and multiple processors to accelerate volume rendering and achieve interactive rendering. The algorithms are based on human visual perception and allow the observer a wide range of mixed render modes. The software was used to reconstruct the pituitary development in zebrafish and observe the degeneration of neurons after injury in a mouse model. Calicum indicator dyes have long been used to study calcium fluxes. We optimized the imaging method to minimize impact on the cell. Live cells were imaged continuously for 45 minutes and subjected to increasing does of a drug. We correlated the amplitude of calcium oscillations to increasing doses of a drug and obtain single cell dose response curves. Because this method is very sensitive and measures single cell responses it has potential in drug discovery and characterization. Microtubules form a dynamic cytoskeleton, which is responsible for cell shape, intracellular transport and has an integral role in mitosis. A hallmark of microtubule organization is lateral interactions. Microtubules are bundles by proteins into dense structures. To estimate the contribution of this bundling process, we created a fractal model of microtubule organization. This model demonstrates that morphology of complex microtubule arrays can be explained by bundling alone. In summary we showed that advances in software for visualization, data analysis and modeling lead to new discoveries.
Oxygen-centered radicals are important intermediates in photobiological, mechanistic and synthetic studies. The majority of precursors of reactive oxyl radicals are labile and thus delicate to handle. Therefore N-(alkoxy)-pyridinethiones and N-(Alkoxy)-thiazolethiones have attracted attention as "mild'' photochemical source of alkoxyl radicals, in the last few years. A disadvantage of the pyridine compounds, is their sensibility to daylight. Despite of their similarities, both molecules behave surprisingly different, if photolyzed in the absence of trapping reagents. The pyridinethione compounds undergo highly efficient radical chain reactions under such conditions while the corresponding thiazolethiones react surprisingly sluggish and give rise to several unwanted side products. The properties of both compounds should be understood and optimized in the frame of this work. Additionally new compounds should be suggested that can also be applied in the photochemical alkoxyl radical generation. Some background information about the generation and application of alkoxyl radicals is provided in chapter 2. Electronic excitations and UV/vis spectroscopy together with a description of quantum chemical approaches that are able to calculate such phenomena are outlined in chapter 3. Chapter 4 deals with the description of the vertical excitation spectra. During the validation CASSCF, CASPT2, TD-DFT and RI-CC2 were tested with respect to their ability to describe the vertical excitations in both compounds. The CASPT2 approach gives accurate descriptions of the electronic excitation spectra of all compounds. The time-dependent DFT results are very sensitive on the choice of the functional and a validation of the results should be always done. On the basis of these computations the spectroscopic visible absorption bands of both compounds were assigned to a pi-->pi* transition in the thiohydroxamic acid functionality. In chapter 5 the mechanism of the thermally and the photochemically induced N,O homolysis in both compounds is unveiled. The near UV-induced N,O homolysis will start from the S2 state. The expected relaxation from the S2- to the S1-state and the dissociation process is expected to be very fast in the case of the thiazolethione compound. The potential surfaces of the pyridine compound in contrast point to a slower N,O bond dissociation. Due to the resulting faster dissociation process the excess energy which results from the photochemical activation is quenched only to small amounts. The maximal possible excess energy of the fragments is lower and a quenching is much more likely in the case of the pyridinethione compounds. This explaines the different reactivities of both compounds. For the also already successfully applied precursor system N-(alkoxy)-pyridineones the computed dissociation paths show courses that clearly predict a slow bond dissociation process. Chapter 6 deals with the tuning of the initial excitation wave length of the known pyridinethiones und thiazolethiones. In the first part the effects of substituents on the thiazolethione heterocycle was examined. The UV/vis spectra of 4 and 5 substituted thiazolethiones can be interpreted like the spectrum of the parent compound. The second part of chapter 6 deals with the identification of a substitution pattern on the pyridine heterocycle which induces a blue shift of the photo active band. The computations showed that electron rich and electron poor substituents result the same effects on the electronic excitation spectra. These substituent effects are additive, but the steric orientation of the substituents has to be taken into account. Chapter 7 describes a computer aided design of new alkoxyl radical precursors. Combining the advantages of both compounds the radical formation should be initiated by an irradiation with light at about 350 nm, and the amount of side products during the radical formation process should be small. To achieve this 18 test candidates were obtained by a systematic variation of the parent compound of the thiazolethione precursor. To identify the promising new precursor systems a screening of the lower electronic excitations of all resulting 18 systems was performed with TD-DFT. For promising systems the N,O or P,O dissociation paths, respectively, were analyzed according to the developed model. N-(methoxy)-azaphospholethione and N-(methoxy)-pyrrolethione seem to be the most promising candidates. The computations predict a strong absorption at about 350 nm respectively 320 nm. Due to the amounts of maximal excess energy and the shapes of the potential surfaces of the N,O bond dissociation paths their reactivity should resemble more the behavior of the pyridinethiones.
Directed cortical actin assembly is the driving force for intercellular adhesion. Vasodilator-stimulated phosphoprotein (VASP) participates in actin-fiber formation and VASP activity is regulated by phosphorylations. We screened for endothelial cell proteins, which bind to VASP dependent on its phosphorylation status. Differential proteomics identified αII-spectrin as novel VASP-interacting protein. αII-spectrin binds to the triple GP5-motif in VASP via its SH3 domain. cAMP-dependent protein kinase-mediated VASP phosphorylation at Ser157 inhibits αII-spectrin/VASP complex formation. VASP becomes dephosphorylated upon formation of cell-cell contacts and in confluent but not in sparse endothelial cells αII-spectrin colocalizes with non-phosphorylated VASP at cell-cell junctions. Ectopic expression of the αII-spectrin SH3 domain fused to claudin-5 translocates VASP to cell-cell contacts and is sufficient to initiate the formation of cortical actin cytoskeletons. αII-spectrin SH3 domain overexpression stabilizes cell-cell contacts and decreases endothelial permeability. Conversely, permeability of VASP-deficient endothelial cells is elevated. In a skin edema model, microvascular leakage is increased in VASP-deficient over wild-type mice. We propose that αII-spectrin/VASP complexes regulate cortical actin cytoskeleton assembly with implications for formation of endothelial cell-cell contacts and regulation of vascular permeability.
Clonidine is an agonist at alpha2-adrenergic receptors that mediate a wide variety of the physiological responses to epinephrine and norepinephrine, such as inhibition of neurotransmitter release as well as sedation and analgesia. As with other therapeutically used alpha2-agonists such as moxonidine and rilmenidine, clonidine possesses an imidazoline structure and is believed to lower blood pressure not only via central and peripheral alpha2-receptors, but perhaps even more so by acting on central “imidazoline I1 receptors” in the brain stem. The molecular structure of these hypothetical “imidazoline I1 receptors” has not yet been identified. In order to test whether ligands with an imidazoline structure elicit pharmacological effects via alpha2-adrenergic receptors or via “imidazoline receptors”, mice were generated with a targeted deletion of all three alpha2-adrenergic receptor subtypes (alpha2ABC-KO). These alpha2ABC-KO mice were an ideal model in which to examine the pharmacological effects of the centrally acting antihypertensives clonidine, moxonidine and rilmenidine in the absence of alpha2-adrenergic receptors. As expected, sedative and analgesic actions of clonidine were completely absent in alpha2ABC-KO mice, confirming the sole role of alpha2-receptors in these properties of clonidine. Clonidine significantly lowered heart rate in anesthetized alpha2ABC-KO and wild-type mice by up to 150 beats/min. A similar bradycardic effect of clonidine was observed in isolated spontaneously beating right atria from alpha2ABC-KO mice. After treatment with the specific If inhibitor ZD 7288, clonidine was no longer able to lower spontaneous beating frequency, suggesting a common site of action. Furthermore, in HEK293 cells stably transfected with HCN2 and HCN4, it could be shown that clonidine inhibits the If current via blockade of pacemaker channels with similar affinity as in isolated alpha2ABC-KO and wild-type atria. This inhibition was demonstrated again in isolated sinoatrial node (SAN) cells from alpha2ABC-KO mice and was identical in potency and efficacy to clonidine inhibition observed in isolated wild-type SAN cells, confirming that inhibition of atrial HCN channels constitutes the alpha2-independent bradycardic action of clonidine. Direct inhibition of cardiac HCN pacemaker channels contributes to the bradycardic effects of clonidine in gene-targeted mice. Thus clonidine-like drugs represent novel structures for future HCN channel inhibitors.
In this thesis we have investigated the effect of NFAT (Nuclear Factor of Activated T Cell) transcription factors on the expression of Rag-(Recombination Activating Genes) genes in murine thymus. The protein products of Rag genes, RAG1 and RAG2, are critical for the recombination and generation of the TCR (T Cell Receptor) repertoire during thymocyte development, and their expression can be suppressed by the activity of NFAT factors. In thymus, the expression of Rag1 and Rag2 genes is induced at the double-negative (DN, CD4-8-) 3 stage, down-regulated at the DN4 stage, re-induced at the double-positive (DP, CD4+8+) stage, and suppressed again at the single-positive (SP, CD4+8- or CD4-8+) stage. Although it is known that TCR signaling suppresses the expression of Rag1 and Rag2 at the SP stage, the signals that mediate the Rag gene down-reulation remain elusive. Here we report that both the calcineurin-NFAT-signaling and MAPKinase signaling pathways, which are activated by TCR signaling during positive selection, mediate the Rag gene down-regulation in DP thymocytes. The calcineurin-NFAT pathway suppresses both the Rag1 and the Rag2 gene expression. This pathway has a stronger suppressive effect on the Rag1 than the Rag2 gene. A synergistic activity between the two NFAT factors NFATc2 and NFATc3 is essential for calcineurin-NFAT signaling to efficiently suppress the Rag gene expression in DP thymocytes. It is likely that the calcineurin-NFAT signaling down-regulates Rag gene expression by suppressing both the Rag anti-silencer element (ASE) activity and the Rag promoter activity. Similarly, MEK-ERK signaling of MAPK signaling pathway mediates the Rag gene suppression in DP thymocytes although the mechanism through which MEK-ERK mediates the Rag gene down-regulation has to be elucidated. In DN thymocytes, it appears that neither the calcineurin-NFAT signaling nor MAPK signaling is involved in the Rag gene down-regulation. However, a role for these two signaling pathways in the Rag gene up-regulation in DN thymocytes is not excluded. In DN thymocytes, pre-TCR signaling stimulates the expression both Nfatc1 and Nfatc2 genes but has no effect on Nfatc3 gene expression. In DN thymocytes, pre-TCR signaling activates Nfatc1α expression but not Nfatc1ß expression, i.e. the two promoters controling Nfatc1 gene xpression are differently controled by pre-TCR signals. Nfatc1α gene expression in DN thymocytes is mainly regulated by the MAPK signaling pathway because activation of Nfatc1α is mediated by MEK-ERK signaling but opposed by JNK signaling. Calcineuirn-NFAT and p38 signaling pathways are not involved in Nfatc1α promoter regulation in DN thymocytes. In DP thymocytes, TCR signaling up-regulates Nfatc1 and Nfatc2 expression but down-regulates Nfatc3 expression. In DP thymocytes, TCR signaling activates Nfatc1α expression. The activation of Nfatc1α in DP thymocytes is mediated by NFATc1, but not or to a less degree by NFATc2 and NFATc3. MEK-ERK, JNK, and p38 signaling pathways are involved in Nfatc1α gene activation in DP thymocytes, probably by activating NFAT trans-activation activity. All these findings illustrate that in thymocytes the expression of NFAT transcription factors – which are essential for thymic development - is controled at multiple levels.
A series of experiments was conducted in order to investigate motor contributions to learning highly skilled action sequences in contrast to sensory contributions. Experiments 1–4 made use of a bimanual-bisequential variant of the serial reaction time task: Presentation of imperative stimuli was arranged such that participants’ left-hand and right-hand responses followed different sequences independently of one another, thus establishing a compound sequence spanning both hands. At least partly independent learning of the two concurrently implemented hand-related sequences was demonstrated after extensive practice under condi-tions of both simultaneous (Experiments 1 & 2) and alternating (Experiments 3 & 4) stimulus presentation and responding. It persisted when there was only one imperative stimulus for presenting both hand-related sequences (Experiments 2–4) instead of two separate imperative stimuli (Experiments 1 & 2), one for each sequence, even when the hand-related sequences were correlated and massive integrated learning of the compound sequence occurred (Ex-periment 4). As for the nature of the independently acquired sequence representations, trans-ferable sequence knowledge was acquired only when there was a separate imperative stimulus for each sequence (Experiments 1 & 2) but not otherwise (Experiments 2–4). The most likely stimulus-based representations which allow for intermanual transfer can be regarded as sen-sory components of highly skilled action sequences, whereas motor components can be con-sidered as being reflected in effector-specific, non-transferable sequence knowledge. The same decomposition logic applies to transferable and non-transferable sequence knowledge observed under conditions of unimanual practice of a single sequence (Experiments 6 & 7). The advantage of practicing a key press sequence with fingers of one hand as opposed to practicing it with fingers of both hands (Experiment 5) also implicates a motor component as the two assignments were equivalent in all other respects. Moreover, Experiments 6 and 7 showed that hand-specific sequence knowledge can develop after relatively little practice (as little as approximately 120 sequence repetitions). Presumably, this occurs especially in tasks with particularly pronounced requirements for coarticulation between consecutive finger movements. In sum, the present series of experiments provides compelling evidence for an effector-specific component of sequence learning. Albeit relatively small in size, it emerged consistently under various conditions. By contributing to the refinement of sequential action execution it can play a role in attaining high levels of performance.
Bacteriosponges contain large amounts of morphologically and phylogenetically diverse microorganisms in their mesohyl. The association is permanent, stable and highly specific, however, little is known about the establishment and maintenance of this association. The first aim of this Ph.D. thesis was to examine cospeciation between eight Aplysina species from the Mediterranean and Caribbean and their cyanobacterial associates. Host phylogeny was constructed with 18S rDNA and ITS-2 sequences using an alignment based on the secondary structure of the molecular markers and five different algorithms each. The genus Aplysina appeared as monophyletic. Aplysina sponges could be distinguished into a Caribbean and a Mediterranean cluster and a possible Tethyan origin is suggested. Comparison of the host phylogeny to the 16S rDNA phylogeny of the cyanobacterial strains revealed the lack of a congruent pattern. Therefore it is proposed that Aplysina sponges have not cospeciated with their cyanobacterial phylotypes and probably also not with other sponge specific microbes. The second aim of this Ph.D. thesis was to examine vertical transmission of microorganisms through reproductive stages of sponges. A general transmission electron microscopy (TEM) suvey revealed a clear correlation in that bacteriosponges always contained many microorganisms in their reproductive stages whereas non-bacteriosponges were always devoid of microbes in their reproductive stages. The transmission of the microbial community via sponge reproductive stages is concluded. Based on the previous results Ircinia felix was chosen for a detailed documentation of vertical transmission. I. felix larvae contained large amounts of microorganisms extracellularly in the central region whereas the outer region was almost free of microbes as shown by TEM. In I. felix juveniles microorganisms were located between densely packed sponge cells. The microbial profiles of I. felix adult, larvae, and juveniles were compared using denaturing gradient gel electrophoresis (DGGE). Similar microbial community patterns were found in adult and the respective larvae indicating that a large subset of the adult microbial community was vertically transmitted. In contrast, microbial communities of larvae pools released by different adult individuals seemed to be more variable. Juvenile banding patterns were a mixture of sponge specific and seawater microbes due to DNA extraction artefacts but demonstrated that at least half of the adult microbial community is present in the next generation. Finally, a comprehensive phylogenetic analysis was conducted by sequencing excised DGGE bands from adult and offspring of the bacteriosponges Agelas wiedenmayeri, I. felix, and Smenospongia aurea and by taking additional 16S rDNA sequences of Ectyoplasia ferox and Xestospongia muta (unpublished data of the laboratory). The identification of 24 vertical transmission clusters in at least 8 eubacterial phyla demonstrates that a complex and uniform microbial community is transferred via sponge reproductive stages. Vertical transmission is specific in that the microorganisms of bacteriosponges, but not those from seawater, are passed on, but unselective in that there appears to be no differentiation between individual sponge-specific lineages. In conclusion, vertical transmission points to a mutualistic and long-term association of bacteriosponges and complex microbial consortia.
The steroid hormones corticosterone/cortisol and aldosterone are synthesized and secreted by the adrenal gland in response to stress or an altered salt-water balance. This is controlled by a negative feedback mechanism referred to as the HPA axis and the RAAS. Actions of these steroid hormones are mediated by the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR), which reside in the cytoplasm in a complex with heat-shock proteins. Both, the GR and the MR belong to the nuclear receptor superfamily and share a common protein structure consisting of three separate domains. However, they have different affinities for various ligands, their actions depend on hormone concentration, they are modulated by pre-receptor mechanisms such as the 11β-HSD2 and they are differently distributed in several tissues. Aldosterone acts via the MR in epithelial and in non-epithelial cells and regulates sodium-water homeostasis, cardiovascular function, neuronal excitability and adipocyte differentiation. So far the analysis of gene inactivation in vivo was limited to mice, but disease models in rats sometimes more closely reflect the situation encountered in humans. Since embryonic stem cells and thus gene targeting in rats is not available, we generated MR knock-down transgenic rats by lentiviral delivery of a shRNA. The F1 progeny of the founder rats showed a wide range of reduced MR mRNA and protein levels in kidney and hippocampus, the two major sites of MR expression. In contrast, expression of the highly homologous GR was unaltered, indicating specificity of gene inactivation. The two MR target genes, Sgk1 and ENaC, were up-regulated while the mRNA levels of other genes such as IK1 and SCD2 was reduced. Similar to the knock-out mice and human patients, the knock-down rats displayed typical signs of pseudohypoaldosteronism type I such as increased serum levels of aldosterone and renin as well as growth retardation. Importantly, we found a linear relationship between MR mRNA expression in kidney, serum aldosterone levels and body weight. Thus, our MR knock-down rats are amongst the first examples of RNAi in vivo and confirm that this technique allows to accomplish graded levels of gene inactivation that mimick human genetic diseases. Secondly, we investigated the role of the GR and the MR for the immunomodulatory activities of glucocorticoids (GCs) in peritoneal macrophages. GCs are involved in the modulation of macrophage function and thereby control the host’s immune responses to pathogens. Therefore, GCs are widely used for the treatment of inflammation and autoimmune diseases. However, concerning these GC activities neither the role of hormone concentration nor the differential contribution of the GR and the MR are known. At first we confirmed that both receptors but not 11β-HSD2 are expressed in peritoneal macrophages. Next, we showed that low levels of corticosterone enhance NO production as well as mRNA expression of pro-inflammatory cytokines, chemokines and enzymes required for mediator synthesis. In contrast, at high corticosterone concentrations macrophage function was strongly repressed. Importantly, inactivation of the GR by lentiviral delivery of siRNAs abrogated both the immunostimulatory and the immunosuppressive GC actions whereas inactivation of the MR had no effect. Furthermore, removal of endogenous GCs by adrenalectomy in vivo induced a pre-activated state in macrophages that could be modulated by corticosterone. We conclude that GCs exert distinct effects on macrophage function dependent on their concentration, and that they act through the GR despite concomitant expression of the MR. In summary, our results confirm that lentiviral delivery of shRNAs is an efficient means to down-regulation gene expression in primary cells and transgenic rats and thereby allows to perform functional studies on gene function that were previously limited to mice.
Inhibition of Nuclear Import of Calcineurin Prevents the Development of Myocardial Hypertrophy
(2007)
The Calcineurin/NFAT signaling cascade is a crucial transducer of cellular function. It has recently been emerged that in addition to the transcription factor NFAT, the phosphatase Calcineurin is also translocated to the nucleus. Our traditional understanding of Calcineurin activation via sustained high Ca2+-levels was also advanced by recent findings from this working group (AG Ritter), which showed that Calcineurin is activated by proteolysis of the C-terminal autoinhibitory domain. This leads to the constitutive activation and nuclear translocation of Calcineurin. Therefore, Calcineurin is not only responsible for dephosphorylating of NFAT in the cytosol thus enabling its nuclear import, its presence in the nucleus is also significant in ensuring the full transcriptional activity of NFAT. Formation of complexes between transcription factors and DNA regulates the transcriptional process. Therefore, the time that transcription factors remain nuclear is a major determinant of transcriptional activity. The movement of proteins over ~40 kDa into and out of the nucleus is governed by the nuclear pore complex (NPC). Transcription factors and enzymes that regulate the activity of these proteins are shuttled across the nuclear envelope by proteins that recognize nuclear localization signals (NLS) and nuclear export signals (NES) within the amino acid sequence of these transcription factors. In this study, the precise mechanisms of Calcineurin nuclear import and export were identified. Additionally to the nuclear localization sequence (NLS) and the nuclear export sequence (NES) within the sequence of Calcineurin, the respective nuclear cargo proteins, responsible for nuclear import, Importinβ1, and for nuclear export, CRM1, were identified. Inhibition of the Calcineurin/importin interaction by a competitive peptide, called Import Blocking Peptide (IBP), which mimicked the Calcineurin NLS, prevented nuclear entry of Calcineurin. A non-inhibitory control peptide showed no effect. Using this approach, it was able to prevent the development of myocardial hypertrophy. In Angiotensin II stimulated cardiomyocytes, both the transcriptional and the translational level was suppressed. Additionally, cell size and expression of Brain natriuretic peptide (as molecular marker for hypertrophy) were significantly reduced compared untreated controls. IBP worked dose-dependent, but did not affect the Calcineurin phosphatase activity. In conclusion, Calcineurin is not only capable of dephosphorylating NFAT, thus enabling its nuclear import, its presence in the nucleus is also important for full NFAT transcriptional activity. Using IBP to prevent the nuclear import of Calcineurin is a completely new approach to prevent the development of myocardial hypertrophy.
Ferromagnetic semiconductors (FS) promise the integration of magnetic memory functionalities and semiconductor information processing into the same material system. The prototypical FS (Ga,Mn)As has become the focus of semiconductor spintronics research over the past years. The spin-orbit mediated coupling of magnetic and semiconductor properties in this material gives rise to many novel transport-related phenomena which can be harnessed for device applications. In this thesis we address challenges faced in the development of an all-semiconductor memory architecture. A starting point for information storage in FS is the knowledge of their detailed magnetic anisotropy. The first part of this thesis concentrates on the investigation of the magnetization behaviour in compressively strained (Ga,Mn)As by electrical means. The angle between current and magnetization is monitored in magnetoresistance(MR) measurements along many in-plane directions using the Anisotropic MR(AMR) or Planar Hall effect(PHE). It is shown, that a full angular set of such measurements displayed in a color coded resistance polar plot can be used to identify and quantitatively determine the symmetry components of the magnetic anisotropy of (Ga,Mn)As at 4 K. We compile such "anisotropy fingerprints" for many (Ga,Mn)As layers from Wuerzburg and other laboratories and find the presence of three symmetry terms in all layers. The biaxial anisotropy term with easy axes along the [100] and [010] crystal direction dominates the magnetic behaviour. An additional uniaxial term with an anisotropy constant of ~10% of the biaxial one has its easy axis along either of the two <110> directions. A second contribution of uniaxial symmetry with easy axis along one of the biaxial easy axes has a strength of only ~1% of the biaxial anisotropy and is therefore barely visible in standard SQUID measurements. An all-electrical writing scheme would be desirable for commercialization. We report on a current assisted magnetization manipulation experiment in a lateral (Ga,Mn)As nanodevice at 4 K (far below Tc). Reading out the large resistance signal from DW that are confined in nanoconstrictions, we demonstrate the current assisted magnetization switching of a small central island through a hole mediated spin transfer from the adjacent leads. One possible non-perturbative read-out scheme for FS memory devices could be the recently discovered Tunneling Anisotropic MagnetoResistance (TAMR) effect. Here we clarify the origin of the large amplification of the TAMR amplitude in a device with an epitaxial GaAs tunnel barrier at low temperatures. We prove with the help of density of states spectroscopy that a thin (Ga,Mn)As injector layer undergoes a metal insulator transition upon a change of the magnetization direction in the layer plane. The two states can be distinguished by their typical power law behaviour in the measured conductance vs voltage tunneling spectra. While all hereto demonstrated (Ga,Mn)As devices inherited their anisotropic magnetic properties from their parent FS layer, more sophisticated FS architectures will require locally defined FS elements of different magnetic anisotropy on the same wafer. We show that shape anisotropy is not applicable in FS because of their low volume magnetization. We present a method to lithographically engineer the magnetic anisotropy of (Ga,Mn)As by submicron patterning. Anisotropic strain relaxation in submicron bar structures (nanobars) and the related deformation of the crystal lattice introduce a new uniaxial anisotropy term in the energy equation. We demonstrate by both SQUID and transport investigations that this lithographically induced uniaxial anisotropy overwrites the intrinsic biaxial anisotropy at all temperatures up to Tc. The final section of the thesis combines all the above into a novel device scheme. We use anisotropy engineering to fabricate two orthogonal, magnetically uniaxial, nanobars which are electrically connected through a constriction. We find that the constriction resistance depends on the relative orientation of the nanobar magnetizations, which can be written by an in-plane magnetic field. This effect can be explained with the AMR effect in connection with the field line patterns in the respective states. The device offers a novel non-volatile information storage scheme and a corresponding non-perturbative read-out method. The read out signal is shown to increase drastically in samples with partly depleted constriction region. This could be shown to originate in a magnetization direction driven metal insulator transition of the material in the constriction region.
Abiotic environmental stress, as evoked by short-term exposure of greenhousegrown plants to ambient ultraviolet radiation (UV), induces chemical and morphological adaptations of plants. Responses depend on the strength of stress and differ between species and tissues of variable age. In two Brassicaceae, Sinapis alba and Nasturtium officinale, stress responses towards short-term exposure to ambient radiation including or excluding UV reveal a high phenotypic plasticity, with strong differences their chemical composition compared to plants that remained in the greenhouse. The most pronounced defensive response against UV, the accumulation of flavonoid pigments, was strongest in young UV-exposed leaves, with an increase of the more effectice flavonol quercetin on the expense of less effectice kaempferol. Glucosinolates and myrosinase enzymes showed highly species-specific responses to UV-stress. Feeding behaviour and larval performance of the oligophagous Brassicaceae specialist, Phaedon cochleariae (Chrysomelidae; Coleoptera) were poorly affected by these differently UV-exposed host plants. Effects of plant stress on larval development were restricted to a minor variation in body mass due to variable food conversion of certain larval instars, which were compensated until pupation. Moreover, larval developmental times were unaffected by UV-exposure, but varied between species and leaves of different age. For P. cochleariae, this lack of variation in larval and pupal development towards UV-altered phytochemistry may suggest a strong genetic fixation of life history traits. In combination, the high plasticity towards variable food quality may correspond to the beetles’s specialisation on a narrow range of chemically highly variable host plants. Apart from being involved in plant defence against generalist herbivores, glucosinolates may also act as recognition cues and feeding stimulants for specialist insects. In earlier studies, glucosinolates were assumed to stimulate feeding by P. cochleariae, and they were suggested to be present on outermost leaf surfaces. However, since these findings were based on crude extraction methods, the presence of feeding stimulants in epicuticular waxes of Brassicaceae was re-investigated. In our study, glucosinolates were not detectable in mechanically removed waxes in Brassica napus and N. officinale, whereas substrate concentrations in solvent leaf extracts corresponded to densities and closure of leaf surface stomata. Therefore, glucosinolates that originate from the mesophyll may have been washed out through open stomata. Neither leaf waxes, nor leaf waxes combined with sinigrin or pure sinigrin evoked feeding. Moreover, in choice tests, these leaf beetles clearly preferred to feed on de-waxed surfaces. Finally, the presence of feeding stimulants in epicuticular waxes is highly unlikely considering the physico-chemical properties of the plant cuticle. The lack of stimulants on the outermost surface corresponds to the plant’s perspective, which should avoid easily accessible feeding stimulants. Nevertheless, the role of glucosinolates for feeding stimulation of P. cochleariae remained unclear. Therefore, S. alba leaf extracts of different polarities were tested in bioassays in order to identify which chemical leaf compounds act as stimulants. In bioassay-guided fractionations of methanol extracts by semi-preparative HPLC, two distinct fractions with stimulating activity were detected, whereas other fractions were not effective. Flavonoids were identified as main component in one stimulating fractions, the second fraction mainly contained glucosinolates, including sinalbin. The combination of both fractions was significantly more stimulating than each individual fraction, indicating additive effects of at least one compound of each fraction. However, since the combined fractions were less effective compared to the original extracts, other compounds may additionally be involved in the complex composition of leaf compounds acting as feeding stimulants for P. cochleariae. Finally, fractionated extracts of UV altered plants were used to test whether the strength of feeding responses depend on different ratios of glucosinolates and flavonoids. However, since the feeding behavior of this leaf beetle was not affected, such quantitative variations were concluded to be less important. The initiation of feeding behaviour may solely depend on the presence of stimulating compounds.
The basic question which drove our whole work was to find a meaningful noncommutative gauge theory even for the time-like case ($\theta^{0 i} \neq 0$). In order to be able to tackle questions regarding unitarity, it is not sufficient to consider theories which include the noncommutative parameter only up to a finite order. The reason is that in order to investigate tree-level unitarity or the optical theorem in loops one has to know the behavior of the noncommutative theory for center-of-mass energies much greater than the noncommutative scale. Therefore an effective theory, that is by construction only valid up to the noncommutative scale, isn't sufficient for our purpose. Our model is based on two fundamental assumptions. The first assumption is given by the commutation relations \eqref{eq:ncalg}. This led to the Moyal-Weyl star-product \eqref{eq:astproduct2} which replaces all point-like products between two fields. The second assumption is to assume that the model built this way is not only invariant under the noncommutative gauge transformation but also under the commutative one. In order to obtain an action of such a model one has to replace the fields by their appropriate \swms. We chose the gauge fixed action \eqref{eq:actioncgf} as the fundamental action of our model. After having constructed the action of the NCQED including the {\swms} we were confronted with the problem of calculating the {\swms} to all orders in $\tMN$. By means of \cite{bbg} we could calculate the {\swms} order by order in the gauge field, where each order in the gauge field contains all orders in the noncommutative parameter (\cf chapter \ref{chapter:swms}). By comparing the maps with the result we obtained from an alternative ansatz \cite{bcpvz}, we realized that already the simplest {\swm} for the gauge field is not unique. In chapter \ref{chapter:ambiguities} we examined this ambiguity, which we could parametrised by an arbitrary function $\astf$. The next step was to derive the Feynman rules for our NCQED. One finds that the propagators remain unchanged so that the free theory is equal to the commutative QED. The fermion-fermion-photon vertex contains not only a phase factor coming from the Moyal-Weyl star-product but also two additional terms which have their origin in the \swms. Beside the 3-photon vertex which is already present in NCQED without {\swms} and which has also additional terms coming from the \swms, too, one has a contact vertex which couples two fermions with two photons. After having derived all the vertices we calculated the pair annihilation scattering process $e^+ e^- \rightarrow \gamma \gamma$ at Born level. By choosing the parameter $\kggg = 1$ (\cf section \ref{sec:represent}), we found that the amplitude of the pair annihilation process becomes equal to the amplitude of the NCQED without \swms. This means that, at least for this process, the NCQED excluding {\swms} is only a special case of NCQED including \swms. On the basis of the pair annihilation process, we afterwards investigated tree-level unitarity. In order to satisfy the tree-level unitarity we had to constrain the arbitrary function $\astf$. We found that the series expansion of $\astf$ has to start with unity. In addition, the even part of the function must not increase faster than $s^{-1/2} \log(s)$ for $s \rightarrow \infty$, whereas the odd part of the $\astf$-function can't be constrained, at least by the process we considered. By assuming these constrains for the $\astf$-function, we could show that tree-level unitarity is satisfied if one incorporates the uncertainties present in the energy and the momenta of the scattered particles, \ie the uncertainties of the center-of-mass energy and the scattering angles. This uncertainties are not exclusively present due to the finite experimental resolution. A delta-like center-of-mass energy as well as delta-like momenta are in general not possible because the scattered particles are never exact plane waves.
Performance Evaluation of Efficient Resource Management Concepts for Next Generation IP Networks
(2007)
Next generation networks (NGNs) must integrate the services of current circuit-switched telephone networks and packet-switched data networks. This convergence towards a unified communication infrastructure necessitates from the high capital expenditures (CAPEX) and operational expenditures (OPEX) due to the coexistence of separate networks for voice and data. In the end, NGNs must offer the same services as these legacy networks and, therefore, they must provide a low-cost packet-switched solution with real-time transport capabilities for telephony and multimedia applications. In addition, NGNs must be fault-tolerant to guarantee user satisfaction and to support business-critical processes also in case of network failures. A key technology for the operation of NGNs is the Internet Protocol (IP) which evolved to a common and well accepted standard for networking in the Internet during the last 25 years. There are two basically different approaches to achieve QoS in IP networks. With capacity overprovisioning (CO), an IP network is equipped with sufficient bandwidth such that network congestion becomes very unlikely and QoS is maintained most of the time. The second option to achieve QoS in IP networks is admission control (AC). AC represents a network-inherent intelligence that admits real-time traffic flows to a single link or an entire network only if enough resources are available such that the requirements on packet loss and delay can be met. Otherwise, the request of a new flow is blocked. This work focuses on resource management and control mechanisms for NGNs, in particular on AC and associated bandwidth allocation methods. The first contribution consists of a new link-oriented AC method called experience-based admission control (EBAC) which is a hybrid approach dealing with the problems inherent to conventional AC mechanisms like parameter-based or measurement-based AC (PBAC/MBAC). PBAC provides good QoS but suffers from poor resource utilization and, vice versa, MBAC uses resources efficiently but is susceptible to QoS violations. Hence, EBAC aims at increasing the resource efficiency while maintaining the QoS which increases the revenues of ISPs and postpones their CAPEX for infrastructure upgrades. To show the advantages of EBAC, we first review today’s AC approaches and then develop the concept of EBAC. EBAC is a simple mechanism that safely overbooks the capacity of a single link to increase its resource utilization. We evaluate the performance of EBAC by its simulation under various traffic conditions. The second contribution concerns dynamic resource allocation in transport networks which implement a specific network admission control (NAC) architecture. In general, the performance of different NAC systems may be evaluated by conventional methods such as call blocking analysis which has often been applied in the context of multi-service asynchronous transfer mode (ATM) networks. However, to yield more practical results than abstract blocking probabilities, we propose a new method to compare different AC approaches by their respective bandwidth requirements. To present our new method for comparing different AC systems, we first give an overview of network resource management (NRM) in general. Then we present the concept of adaptive bandwidth allocation (ABA) in capacity tunnels and illustrate the analytical performance evaluation framework to compare different AC systems by their capacity requirements. Different network characteristics influence the performance of ABA. Therefore, the impact of various traffic demand models and tunnel implementations, and the influence of resilience requirements is investigated. In conclusion, the resources in NGNs must be exclusively dedicated to admitted traffic to guarantee QoS. For that purpose, robust and efficient concepts for NRM are required to control the requested bandwidth with regard to the available transmission capacity. Sophisticated AC will be a key function for NRM in NGNs and, therefore, efficient resource management concepts like experience-based admission control and adaptive bandwidth allocation for admission-controlled capacity tunnels, as presented in this work are appealing for NGN solutions.
In 2001 the 433 m deep Messel 2001 borehole was drilled in the centre of the Messel Pit, 25 km south of Frankfurt (Germany). Geoscientific results from this drilling clarified the origin of the circular-shaped basin as a maar-diatreme-structure. Recovered deposits consist of lacustrine sediments (0-240 m) and volcaniclastic rocks such as lapilli tuffs (240-373 m) as well as rocks of the underlying diatreme breccia (373 433 m). The lapilli tuffs, as main interest here, show little differentiation on a macro- and microscopic scale and appear as a massive and unsorted volcaniclastic body with dominating juvenile lapilli and accidental clasts mostly in the range of (sub)millimetres to centimetres in diameter. This study presents rock magnetic properties measured on core samples of the volcaniclastic units and explains the origin of downhole magnetic anomalies detected during the drilling project in 2001. Magnetic behaviour of the erupted material is related to fine-grained, Fe-rich (titano)-magnetites, which are dispersed within the juvenile lapilli. Temperature-dependent susceptibility experiments, isothermal remanent magnetisation and hysteresis investigations demonstrate similar ferrimagnetic properties throughout the volcaniclastic material, in terms of composition, coercivity and grain size (pseudo-single-domain particles) of the ferrimagnetic minerals. Thus, during emplacement of the erupted material, the ferrimagnetic minerals had the same remanence acquisition potential. However, demagnetisation experiments show different magnetic stability behaviour of the acquired natural remanent magnetisation (NRM). Heating experiments prove the acquisition of thermal remanent magnetisation (TRM) dominated by temperature effects which could have been occurred during eruption and deposition of volcanic material, forming the Messel maar-diatreme. It is assumed that the upper half of the lapilli tuffs was deposited at relatively low depositional temperatures (<300 °C), whereas the material of the lower half took advantage of higher temperatures (>>300 °C). To understand the rock magnetic character within the Messel maar-diatreme-facies, particle grain sizes, the degree of the relative fraction dominance and the shape of the juvenile fragments have been studied in more detail. Image analytical methods as well as major and trace element analyses on the juvenile fraction support the clear subdivision of the lapilli tuffs. These findings in combination with rockmagnetic data indicate a separation into a relatively hot, geochemically undifferentiated eruption phase and a colder, differentiated phase. A two-condition eruption stage at the end of the Messel volcanic activity is suggested. The juvenile particles account for the temperature evolution and heat conditions during deposition of the Messel tuffs and contribute to the origin of magnetic field anomalies. Based on gravity parameters and the results of magnetisation properties, the potential field 3D-model of the Messel subsurface explains the negative ground anomalies, calculates the mass and volume parameters of the drilled lithozones and shows the asymmetric appearance of the diatreme-structure.
NO has been described as an important component involved in the development of the hypersensitive reaction (Delledonne et.al., 1998). Furthermore, NO induces expression of a set of defence gene, such as PR-1, PAL1 and chalcone synthase (CHS), and accumulation of SA (Durner et al., 1998). In this study, transgenic plants with altered NO levels were used to study the role of NO in plant defence. Arabidopsis plants which, due to expression of a bacterial NO dioxygenase, exhibit lower levels of NO than wild-type plants, show several weakened defence response, including the oxidative burst and expression of phenylpropanoid pathway genes. By contrast, constitutive expression of a bacterial NO synthase in Arabisopsis results in increased levels of endogenous NO. However, these plants do not show constitutively activated defence responses, but suffer from increased susceptibility to various strains of P. syringae. This might indicate that a gradient in NO production rather than constitutive elevation of NO is necessary to trigger plant defence responses. Nevertheless, NO seems to be important for regulation of the oxidative state in plant cells. This function of NO is important during leaf senescence. The data of the present work indicate that NO acts as senescence-delaying factor during plant development. The molecular action of NO in plants and signalling cascades in which NO is involved as second messenger are still poorly understood. Experiments addressing the selective quantification of NO in intact plant tissue, the identification of NO-target proteins as well as the function of NO-modified biomolecules might help to understand the role of NO in plants. Non-host resistance consists of several layers of defence that include preformed compounds existing in plants before pathogen infection and induced defences which the plant activates after recognition of a pathogen. The role of inducible defences in preventing multiplication of non-adapted bacteria is not clear. Our experiments suggest that to restrict non-adapted bacterial growth, pre-formed antimicrobial compounds and an early inducible cell wall-based defence might play an important role in Arabidopsis leaves. Upon inoculation with non-adapted bacteria, we have observed early, TTSS-independent up-regulation of PAL1 and BCB, two lignin biosynthesis genes which might be involved in papilla formation or other kinds of cell wall fortification. Moreover, Arabidopsis pal1 knockout lines permit significantly higher survival of non-adapted bacteria in leaves than wild-type plants, suggesting a functional importance of PAL1 up-regulation. Although non-host bacteria, like host bacteria, induce accumulation of SA and PR gene expression in a TTSS-dependent manner, SA-dependent or JA/ET-dependent defences do not directly contribute to non-host resistance. Moreover, non-adapted bacteria activate similar defence signalling pathways as do host bacteria. However, because of varieties in effector protein composition between different non-adapted bacterial strains, the activated signalling pathways might also include different compounds. The Arabidopsis ecotype Ler 0 is more susceptible to a non-adapted strain of P. syringae than ecotype Col-0. Although differences in glucosinolate content and composition between those ecotypes exist, they are probably not a major reason for the observed difference in non-host resistance. To further understand the mechanisms underlying non-host resistance, the generation of double or triple mutants with deficits in both cell wall-based defences and SA-dependent signal cascades is necessary. Moreover, the study of genome polymorphism and composition of secondary metabolites between Ler-0 and Col-0 can shed new light into the mechanisms of non-host resistance against bacterial pathogens. Additionally, experiments addressing papilla formation and callose biosynthesis in Ler-0 and Col-0 could help to further elucidate bacterial non-host resistance. Our data indicate that localized contact of Arabidopsis leaves with non-adapted bacteria, type III secretion-defective P. syringae strains and bacterial pathogen-associated molecular patterns (PAMPs) induce systemic acquired resistance (SAR) at the whole plant level. This finding contrasts the general belief that an HR or other leaf necroses are required for SAR induction. The observed symptomless systemic response was abolished in all SAR-deficient mutants tested in this study, but was intact in the jar1 mutant, which is compromised in induction of ISR, indicating that non-host bacteria and PAMPs induce SAR in a mechanistically similar way than host bacteria. In addition, our data show that the extent of SA accumulation or PR gene expression induced at sites of virulent or avirulent P. syringae inoculation rather than the amount of tissue necroses or jasmonate accumulation determine the magnitude of SAR. The fact that systemic responses were also triggered after local treatment with type III secretion-defective P. syringae strains and bacterial PAMPs indicate that induction of SAR is TTSS-independent. Instead, recognition of general elicitors like flagellin and LPS play an important role in activation of the SAR process. To broaden the concept of PAMP-based SAR initiation, further general elicitors from bacteria and fungal pathogens should be tested for their capability to induce SAR. Screens for mutants with deficiency in SAR activation by individual PAMPs can help to identify new components involved in the SAR signalling cascade. Possible functions of PAMPs as mobile systemic signals should be tested in future experiments. By selection of candidate genes whose expression is up-regulated in Arabidopsis leaves infected with avirulent and virulent P. syringae and pathophysiological analyses of corresponding T-DNA knockout lines, FLAVIN-DEPENDENT MONOOXYGENASE1 (FMO1) was identified as a key SAR regulator. SAR triggered by P. syringae is completely abolished in fmo1 mutant plants, and pathogen-induced expression of FMO1 in systemic leaves is closely correlated with the capability of different Arabidopsis lines to develop SAR. According to our findings, we have proposed that the FMO1 acts in signal amplification in non-inoculated, systemic leaves to trigger SAR. Experimental verification of the postulated potential amplification cycle underlying SAR should be tested in future experiments. The generation of transgenic lines expressing FMO1::GFP will provide useful information about the cellular localization of the FMO1 protein. Moreover, a comparative metabolomic analysis using SAR-induced wild-type, fmo1 knockout and FMO1 overexpressing lines can be used to identify substrates and reaction products of the FMO1 monooxygenase. As the single yeast FMO (yFMO) provides oxidizing equivalents at the ER for correct protein folding, expression of FMO1 in yfmo mutant yeast combined with protein activity assays might indicate whether FMO1 exhibits functional similarities with yeast FMO, e.g. in assuring proper folding of ER-targeted proteins essential for SAR establishment. Identification of further genes involved in activation of systemic resistance and biochemical characterization of the corresponding proteins can help to understand the SAR process in more detail.
This thesis deals with the isolation and structural elucidation of bioactive naphthylisoquinoline alkaloids and related analogs. The mode of action of the antiplasmodial activity exhibited by the naphthylisoquinoline alkaloids was explored and compared to that of the antimalarial drug chloroquine. Furthermore, the phase 1 and 2 metabolism of dioncophyllines A and C and dioncopeltine A were investigated. In detail the following results have been obtained: • From the leaves of the recently discovered East African liana A. tanzaniensis six naphthylisoquinoline alkaloids were isolated. • The leaves of a botanical yet undescribed Ancistrocladus species, collected by Prof. Dr. V. Mudogo in the Democratic Republic of Congo in the habitat Yeteto near the town Ikela, were analyzed for naphthylisoquinoline alkaloids for the first time. The isolation work led to the first identification of an N,C-coupled naphthyldihydroisoquinoline alkaloid; ancistrocladinium B. Phytochemical investigation of the roots of the Congolese Ancistrocladus species (habitat Yeteto), , afforded five new derivatives of known naphthylisoquinoline alkaloids, namely 5'-O-demethylhamatine, 5'-O-demethylhamatinine, 6-O-demethylancistroealaine A, 6,5'-O,O-didemethylancistroealaine A, and 5-epi-6-O-methylancistrobertsonine A, along with six known naphthylisoquinoline alkaloids. • The antiplasmodial activity guided purification of 60Co irradiated samples containing commercially available naphthylisoquinoline related substances, afforded the isolation of the irradiation products 3,4-dihydro-1-isoquinolinone, 3,4-dihydro-1-isoquinolineamine, and 1,2,3,4-tetrahydro-1,2-diazirino-isoquinoline. The compounds were found to be more active than the starting material, although only exhibiting weak antiplasmodial activity against P. falciparum. • The effect on the absorption spectrum of FPIX due to complex formation with the naphthylisoquinoline alkaloids dioncophyllines A and C, dioncopeltine A korupensamine A, and ancistrocladine was examined by a titration study. Job's plot analyses by UV-spectroscopy determined the stoichiometry for the complex formation of FPIX and naphthylisoquinoline alkaloids to be 2:1. Furthermore, the dissociation constants for the complexation with FPIX were determined for each of the naphthylisoquinoline alkaloids investigated. Dioncophylline C and dioncopeltine A were found to possess dissociation constants, which are comparable to the one reported for the antimalarial drug chloroquine. The ability of ESI to transfer noncovalent solution-phase assemblies intact into the gas phase, was conducted on solution mixtures of naphthylisoquinoline alkaloid and FPIX, as well as on mixtures of chloroquine and FPIX. The mass spectrometry analyses revealed several peaks, which corresponded to the complex formation of FPIX to the respective ligands investigated. The most interesting results obtained were the detection of peaks corresponding to the complex formation between a chelated dimer of FPIX and dioncophylline Cand of peaks corresponding to a double protonated tetramer of FPIX – consisting of two chelated -oxo dimers of FPIX – in complex formation with two molecules of chloroquine. • Two phase 1 metabolism products of dioncophylline A were identified. Coelution in combination with HPLC-MS/MS, NMR, and CD investigations assigned the major metabolic product as 5'-O-demethyldioncophylline A. The minor metabolic product was only present in small amounts, which disabled an unambiguous structural characterization of the compound. However, as deduced from the mass spectrometry analyses and exclusion of a possible metabolic oxidation product by coelution with authentic reference material, the metabolite should possess a 4-hydroxylated isoquinoline portion and is assumed to be represented by structure. Dioncophylline C and dioncopeltine A were found to be stable to phase 1 metabolism reactions caused by rat liver microsomes.
Since the fruit fly Drosophila melanogaster entered the laboratories as a model organism, new genetic, physiological, molecular and behavioral techniques for the functional analysis of the brain rapidly accumulated. Nowadays this concerted assault obtains its main thrust form Gal4 expression patterns that can be visualized and provide the means for manipulating -in unrestrained animals- groups of neurons of the brain. To take advantage of these patterns one needs to know their anatomy. This thesis describes the Virtual Insect Brain (VIB) protocol, a software package for the quantitative assessment, comparison, and presentation of neuroanatomical data. It is based on the 3D-reconstruction and visualization software Amira (Mercury Inc.). Its main part is a standardization procedure which aligns individual 3D images (series of virtual sections obtained by confocal microscopy) to a common coordinate system and computes average intensities for each voxel (volume pixel). The VIB protocol facilitates direct comparison of gene expression patterns and describes their interindividual variability. It provides volumetry of brain regions and helps to characterize the phenotypes of brain structure mutants. Using the VIB protocol does not require any programming skills since all operations are carried out at a (near to) self-explanatory graphical user interface. Although the VIB protocol has been developed for the standardization of Drosophila neuroanatomy, the program structure can be used for the standardization of other 3D structures as well. Standardizing brains and gene expression patterns is a new approach to biological shape and its variability. Using the VIB protocol consequently may help to integrate knowledge on the correlation of form and function of the insect brain. The VIB protocol provides a first set of tools supporting this endeavor in Drosophila. The software is freely available at http://www.neurofly.de.
The insulin receptor ortholog EmIR of the fox-tapeworm Echinococcus multilocularis displays significant structural homology to the human insulin receptor (HIR) and has been suggested to be involved in insulin sensing mechanisms of the parasite’s metacestode larval stage. In the present work, the effects of host insulin on Echinococcus metacestode vesicles and the proposed interaction between EmIR and mammalian insulin have been studied using biochemical and cell-biological approaches. Human insulin, exogenously added to in vitro cultivated parasite larvae, (i) significantly stimulated parasite survival and growth, (ii) induced DNA de novo synthesis in Echinococcus, (iii) affected overall protein phosphorylation in the parasite, and (iv) specifically induced the phosphorylation of the parasite’s Erk-like MAP kinase orthologue EmMPK1. These results clearly indicated that Echinococcus metacestode vesicles are able to sense exogenous host insulin which induces a mitogenic response. To investigate whether EmIR mediates these effects, anti-EmIR antibodies were produced and utilized in biochemical assays and immunohistochemical analyses. EmIR was shown to be expressed in the germinal layer of the parasite both on the surface of glycogen storing cells and undifferentiated germinal cells. Upon addition of exogenous insulin to metacestode vesicles, the phosphorylation of EmIR was significantly induced, an effect which was suppressed in the presence of specific inhibitors of insulin receptor-like tyrosine kinases. Furthermore, upon expression of EmIR/HIR receptor chimera containing the extracellular ligand binding domain of EmIR in HEK 293 cells, a specific autophosphorylation of the chimera could be induced through the addition of exogenous insulin. These results indicated the capability of EmIR to sense and to transmit host insulin signals to the Echinococcus signaling machinery. The importance of insulin signaling mechanisms for parasite survival and growth were underscored by in vitro cultivation experiments in which the addition of an inhibitor of insulin receptor tyrosine kinases led to vesicle degradation and death. Based on the above outlined molecular data on the interaction between EmIR and mammalian insulin, the parasite’s insulin receptor orthologue most probably mediates the insulin effects on parasite growth and is, therefore, a potential candidate factor for host-parasite communication via evolutionary conserved pathways. In a final set of experiments, signaling mechanisms that act downstream of EmIR have been analyzed. These studies revealed significant differences between insulin signaling in Echinococcus and the related cestode parasite Taenia solium. These differences could be associated with differences in the organo-tropism of both species.
Pathogenic relevance of autoantibodies to type XVII collagen from pemphigoid gestationis patients
(2007)
Pemphigoid gestationis (PG) and bullous pemphigoid (BP) are subepidermal autoimmune blistering diseases characterized by self-reactive T and B cells specific for the transmembrane hemidesmosomal protein type XVII collagen/BP180. Major T and B cell epitopes are located within the immunodominant 16th non-collagenous domain A (NC16A) of type XVII collagen. It has been suggested that pathogenically relevant autoantibodies also bind to this immunodominant region. The aim of this study was to map the epitopes targeted by blister-inducing human autoantibodies. For this purpose, we used an in vitro model of autoantibody-induced leucocyte-dependent dermal-epidermal separation. In contrast to the majority of patients with BP (7 of 10), preadsorption against a recombinant form of the NC16A region abolished the blister-inducing potential of autoantibodies from all PG patients tested (n=5). Using overlapping synthetic peptides, we demonstrate that PG autoantibodies bind to 2 defined epitopes within the NC16A region (aa 500-514 and aa 511-523). Preadsorption using an affinity matrix containing these two epitopes completely abolished dermal-epidermal separation induced by PG autoantibodies (in 8 of 9 patients). These findings provide new insights into the pathogenesis of pemphigoid diseases and should prove helpful for the development of an antigen-specific immunoadsorption therapy in PG.
The aim of the present study was to design different dosage forms as carrier systems to deliver sorafenib to the lung of BXB-23 transgenic mice using different routes of administration. Three dosage forms were used one of them was an oil-in-water emulsion and the oral route was chosen for this experiment. The other delivery system was a liposome preparation for intratracheal instillation. In this case the oral route was considered as a control experiment. The last dosage form was PLGA microspheres. Before sorafenib administration it was important to develop a HPLC method to assess sorafenib absorption after its administration and to determine its concentrations in mouse serum. The HPLC method allowed sorafenib quantification in small volumes (30 µl) of mouse serum and tissues. The developed HPLC method was validated resulting in satisfactory selectivity, good linearity, good accuracy and precision over the concentration range examined. Sorafenib was successfully incorporated in a fat emulsion (o/w) using a traditional method resulting in a white homogenous emulsion and no particle aggregation was observed. Sorafenib exhibited antitumor activity on the lung adenoma in BXB-23 transgenic mice when administered orally (2 mg sorafenib per mouse) in the emulsion preparation. The determined effect was an approximately 29 % reduction in the tumor area of the adenoma foci and a proliferation reduction. In order to improve the pharmacological effects of sorafenib on the lung adenoma in BXB-23 mice, the targeting of sorafenib directly to the site of action (the lung) was an attractive concept. For this purpose the intratracheal route was used. Since sorafenib administration by instillation required incorporation of sorafenib in a dosage form suitable for its lipophilic nature, a liposome suspension was the second dosage form used. A lyophilization method was employed for sorafenib liposome preparation utilizing dilauroylphosphatidylcholine (DLPC) which is safe and tolerable for the lung. Incorporation of sorafenib in the liposomes did not influence the particle size and its distribution. The sorafenib liposomes showed high encapsulation efficiency, good stability at 4 °C for one month and satisfactory in vitro release properties and inhibited Raf-1 mediated activation of ERK in cell culture assay. In a pharmacokinetic experiment sorafenib loaded liposomes were instilled directly into the lung. The results revealed that a significant level of sorafenib was achieved in the lung tissues after 2 hours and then reduced after 48 h and remained nearly constant for one week. On the other hand, only traces of sorafenib were found in the mice serum up to 48 h. Subsequently, the pharmacological activity of sorafenib (1 mg per mouse) was studied when delivered in a liposomal suspension intratracheally to treat the lung adenoma of BXB-23 mice. The data of this experiment demonstrated that sorafenib intratracheal instillation resulted in a reduction of tumor area of adenoma foci (67 %) and an elevation of the percent of apoptotic cells. In contrast, prolongation of the treatment period did not further enhance sorafenib activity on the lung adenoma. This previous finding suggested a development of multidrug resistance (MDR) by the adenoma foci cells against sorafenib instillation, which was examined by immunohistochemistry staining. The percent of MDR positive cells was higher after two and three weeks sorafenib liposome instillation treatment than that after one week treatment. The last dosage form used for sorafenib was microspheres, which were prepared by emulsion-diffusion-evaporation method using biodegradable PLGA 50:50 resulting in a white lyophilized powder. The system was characterized physicochemically and revealed a good microspheres yield, high encapsulation efficiency, a homogenous particle size distribution and slow in vitro release of sorafenib. The other strategy studied in the present research project was gene delivery to target the lung bearing tumor of BXB-23 mice using a non-viral vector (polyethylenimine). Polyethylenimine (PEI) was used to investigate its efficiency in transfecting lung bearing tumor of BXB-23 mice model and its ability to transfect the adenoma foci cells. LacZ, which encodes Beta-galactosidase was used in the present study as a reporter gene and was complexed with PEI before delivered intravenously. A high LacZ expression in the alveolar region with some expression in the adenoma foci was observed. On contrary, a low LacZ expression in the alveoli and in the adenoma foci was achieved after instillation of the same polyplex intratracheally.
In physiological conditions platelets have a major role in maintaining haemostasis. Platelets prevent bleeding from wounds by distinguishing normal endothelial cells in vasculature from areas with lesions to which they adhere. Interaction of platelet agonists and their receptors is controlled by intracellular signaling molecules that regulate the activation state of platelets. Very important intracellular signaling molecules are cyclic nucleotides (cGMP and cAMP), both involved in inhibition of platelet activation. Formation of cGMP and cAMP in platelets is stimulated by endothelial-derived NO and prostacyclin (PGI2), which then mediate inhibition of platelets by activating protein kinase G (PKG) and protein kinase A (PKA). Recently, it has been suggested that reactive oxygen species (ROS) represent new modulators of cell signaling within different cell types. The work summarized here describes the involvement of platelet ROS production in platelet activation, the relation of NO/cGMP/PKG I pathway to ROS and to mitogen-activated protein kinases (MAP kinase) signaling, and the involvement of cyclic nucleotides in megakaryocyte and platelet development. Platelets activated with different agonists produce intracellular but not extracellular ROS by activation of NAD(P)H oxidase. In addition, ROS produced in platelets significantly affects αIIbβ3 integrin activation but not alpha/dense granule secretion and platelet shape change. Thrombin induced integrin αIIbβ3 activation is significantly decreased after pretreatment of platelets with NAD(P)H oxidase inhibitors and superoxide scavengers. These inhibitors also reduce platelet aggregation and thrombus formation on collagen under high shear and achieve their effects independently of the NO/cGMP pathway. ADP secreted from platelet dense granules with subsequent activation of P2Y12 receptors as well as thromboxane A2 release are found to be important upstream mediators of p38 MAP kinase activation by thrombin. However, p38 MAP kinase activation does not significantly contribute to calcium mobilization, P-selectin expression, αIIbβ3 integrin activation and aggregation of human platelets in response to thrombin. Finally, PKG activation does not stimulate, but rather inhibit, p38 and ERK MAP kinases in human platelets. Further study revealed that cyclic nucleotides not only inhibit platelet activation, but are also involved, albeit differentially, in megakaryocyte and platelet development. cAMP is engaged in haematopoietic stem cell differentiation to megakaryocytes, and cGMP has no impact on this process. While PKA is already present in stem cells, expression of proteins involved in cGMP signaling (soluble guanylyl cyclase, sGC; PKG) increases with maturation of megakaryocytes. In the final step of megakaryocyte maturation that includes release of platelets, cGMP and cAMP have mild but opposing effects: cGMP increases platelet production while cAMP decreases it indicating a finely regulated process that could depend on stimulus coming from adjacent endothelial cells of sinusoids in bone marrow. The results of this thesis contribute to a better understanding of platelet regulation and of the possible molecular mechanisms involved in megakaryocyte maturation in bone marrow vascular microenvironment.
This volume brings together several authors from different areas of psychology and the neighbouring social sciences. Each one contributes their own perspective on the growing interest topic of subjective well-being. The aim of the volume is to present these divergent perspectives and to foster communication between the different areas. Split into three parts, this volume initially discusses the general perspectives of subjective well-being and addresses fundamental questions, secondly it discusses the dynamics of subjective well-being and more specific research issues to give a better understanding of the general phenomenon, and thirdly the book emphasizes the social context in which people experience and report their happiness and satisfaction. The book will be of great interest to social and clinical psychologists, students of psychology and sociology and health professionals.
Integrins are transmembrane receptors transmitting mechanical signals from the extracellular matrix (ECM) to the cytoskeleton (outside-in-signaling). Many molecular defects in the link between cytoskeleton and ECM are known to induce cardiomyopathies. alpha v integrin appears to play a major role in several processes relevant to remodeling, such as binding and activation of matrix metalloproteinases as well as regulation of cell proliferation, migration, and differentiation. We hypothesized that alpha v integrin-mediated signaling is required for the compensatory hypertrophy after aortic banding (AB) and associated with the modulation of ECM protein expression. Mice were treated in vivo with a specific integrin alpha v inhibitor or vehicle via osmotic minipumps starting 1 day prior to aortic banding (AB). At day 2 and day 7 following AB or sham-operation, the mice were examined by echocardiography and hemodynamic analyses were performed. Treatment of alpha v Integrin inhibitor led to a dilated cardiomyopathy and congestive heart failure in AB mice (dilated left ventricle, depressed LV function, and pulmonary congestion), but not to hypertrophy as observed in mice without inhibitor treatment. Investigation of downstream signaling revealed significant activation of the p38 Mitogen-Activated Protein Kinase (MAPK), the Extracellular signal-Regulated Kinases 1 and 2 (Erk 1/2), Focal Adhesion Kinase (FAK) and tyrosine-phosphorylation of c-Src in mice 7 days after AB. This response was blunted in mice treated with integrin alpha v inhibitor. Microarrays probing for a total of 96 cell adhesion and ECM genes identified various genomic targets of integrin alpha v mediated signalling. 7 days after AB 18 ECM genes were up-regulated more than 2-fold (n=6), e.g. collagen (8.11 ± 2.2), fibronectin (2.32 ± 0.94), secreted protein, acidic and rich in cysteine (SPARC, 3.78 ± 0.12), A disintegrin-like and metalloprotease (reprolysin type) with trombospondin type 1 (Adamts-1, 3.51 ± 0.81) and Tissue inhibitor of metalloproteinase 2 (TIMP2, 2.23 ± 0.98), whereas this up-regulation was abolished in mice that were treatd by integrin alpha v inhibitor via mini pumps. We conclude that signaling downstream of integrin alpha v is mediated by the MAPK, FAK and c-Src pathways leading to an up-regulation of extracelluar matrix components necessary for the compensatory response of the heart under a condition of pressure overload.